System and Method for Forming Bone, Ligament, and Bone-Ligament Constructs

ABSTRACT

A system and method for forming a bone construct include providing bone marrow stromal cells on a substrate without disposing the cells within an exogenous scaffold, and culturing the cells in vitro in osteogenic media such that the cells form a confluent monolayer and detach from the substrate to form a self-organized three-dimensional bone construct. A system and method for forming a ligament construct using fibrogenic media and a system and method for forming a functionally integrated bone-ligament construct are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 60/899,178 filed Feb. 2, 2007, which is incorporated by referenceherein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to tissue engineering, and more particularly to asystem and method for producing engineered bone, ligament, andbone-ligament constructs.

2. Background Art

Bone is a vascularized tissue composed of a number of different types ofcells. The tissue is predominately made up of a mineralized type Icollagen matrix, and crystals within the mineralized matrix are composedof hydroxyapatite, a form of calcium phosphate. The three differenttypes of cells in bone are osteoblasts, osteocytes, and osteoclasts.These cells each have different functions that allow bone tissue tocontinually remodel itself. Osteoblasts are the cells involved in thedeposition and mineralization of type I collagen. These cells are roundin morphology with cytoplasmic projections. Once the osteoblast is fullysurrounded by mineralized matrix, it differentiates into an osteocyte.Osteocytes are cells that sit in open lacunae within mineralized bone.The functions of an osteocyte are to both resorb bone and to deposit newbone. Osteocytes are connected to other cells via cytoplasmicprojections that can travel through channels within the mineralizedmatrix. Finally, osteoclasts are large multinucleated cells with largevacuoles that are involved in the bone resorption. Osteoclasts have twodifferent types of plasma membranes: clear zones and ruffled borders.The ability of bone to remodel itself allows it to change itsarchitecture and constitution (e.g. local density) with changes in itsloading environment. Also, when fractured or inflicted with a smalldefect, bone can easily heal by the combination of processes of collagendeposition, mineralization, and resorption.

Ligaments are dense, relatively avascular connective tissues of themusculoskeletal system that help control joint motion, along withmuscle. These tissues connect one bone to another and function toprovide mechanical stability in joints, serve as a guide to jointmotion, and prevent excess motion. About 80% by volume of ligamenttissue is composed of longitudinally aligned collagen bundles. Most ofthe collagen is type I, however, type III is also present, as iselastin. Fibroblasts are the cellular component in ligaments and make upapproximately 20% of the adult tissue volume. These cells attach to theindividual collagen bundles and are elongated longitudinally.

The interface between bone and ligament is referred to as an enthesis.The purpose of the enthesis tissue is to transmit loads with highfidelity over a minimal volume of tissue from the compliant ligament tothe stiff bone at the bone-ligament interface. This tissue is composedof four different zones that aid in the transition between the twovastly different tissues. The four zones of the enthesis are ligament,unmineralized fibrocartilage, mineralized fibrocartilage, and bone. Thetransition from ligament to unmineralized fibrocartilage is gradual,whereas a distinct boundary exists between unmineralized and mineralizedfibrocartilage in adult tissue. This boundary is termed a tidemark andcan be identified using hematoxylin and eosin (H and E) staining due toits extreme basophilic nature (Claudepierre and Voisin, Joint Bone Spine72: 32, 2005; Benjamin et al., J Anat 208: 471, 2006). Fibrocartilagezones are composed of type II collagen and proteoglycans such asaggrecan, biglycan and decorin. The cells in fibrocartilage have thephenotype of chondrocytes, round and arranged in pairs or rows withinlacunae. There are no molecular markers that are unique to this type oftissue, however, fibrocartilage, and in general the enthesis, isgenerally characterized by the presence of type II collagen due to thefact that this protein is not present in the neighboring ligament andbone tissues (Waggett et al., Matrix Biol 16: 457, 1998).

There are approximately one million surgeries each year in the UnitedStates that require bone and ligament grafts to replace tissue damagedby disease or extensive trauma. Several limitations are associated withgrafting, such as graft availability, donor site morbidity, and immunerejection. Because of these complications, strategies are beingdeveloped to engineer bone and ligament tissue in vitro.

Current approaches to engineer bone and ligament involve the design of athree-dimensional (3D) scaffold that promote the differentiation andproliferation of osteogenic or fibroblastic cells and the deposition andmineralization of an osteogenic or fibroblastic extracellular matrix(ECM). The scaffold design rubrics also include the ability to withstandphysiological loads in vivo and either the eventual incorporation intothe native tissue or degradation during the course of tissue development(Salgado et al., Macromol Biosci 4: 743, 2004). Polymers such aspoly(lactic-co-glycolic acid), poly(propylene fumarates), andpoly(caprolactones) provide a matrix that promotes cell adhesion andmigration, allow for the deposition and mineralization of ostcogcnic ECMin vitro, and have predictable degradation rates, but lack themechanical properties needed to withstand the loads placed on naturalbone in vivo (Ishaug et al, J Biomed Mater Res 36: 17, 1997; Vehof etal., J Biomed Mater Res 60: 241, 2002; Peter et al., J Biomed Mater Res43: 422, 1998).

Hydroxyapatite and b-tricalcium phosphates are ceramics used for bonescaffolding that also promote cell adhesion and proliferation and, whenimplanted, have shown positive results in regards to bone regenerationin vivo. However, the brittle nature of ceramics inhibits their use inhealing large defects (Salgado, 2004; Ducheyne and Qiu, Biomaterials 20:2287, 1999). Polymer-ceramic composite scaffolds such as calciumphosphate salts embedded in poly(caprolactones) have been designed tomitigate the problems with using each material alone, but a significantpercentage of cells fails to attach to the composite scaffold due tolimited surface-to-volume ratio (Zhou et al., Polym Int 56: 333, 2007;Zhou et al., Biomaterials 28: 814, 2007). Single layer cell sheets grownfrom bone marrow stromal cells (BMSC) and wrapped around compositescaffolds have recently been shown to form constructs that resemble bonein vitro and in vivo (Zhou et al., Biomaterials 2007). However, thismethod still involves the use of an exogenous scaffolding that mustincorporate into native tissue. Therefore, while scaffolding strategiesappear to promote osteogenic or fibroblastic cell growth, limitationssuch as immune rejection, degradation, and nonphysiological mechanicalproperties of the scaffold need to be considered when used for bone andligament repair.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a photograph of an engineered bone construct (EBC) accordingto the present invention 7 days post 3D construct formation (scale bar=5mm);

FIGS. 1 b-1 e depict staining of an EBC according to the presentinvention for (b) hematoxylin and eosin; (c) alkaline phosphatase; (d)alizarin red for calcification; and (e) collagen I (scale bars=250 mm);

FIGS. 2 a-c illustrate transmission electron microscopy (TEM) of EBC invitro, wherein FIG. 2 a depicts an osteoblast in an EBC havingcytoplasmic projections (arrows) and surrounded by clusters of mineral(*) (scale bar=1.5 mm); FIG. 2 b illustrates mineral clusters (*) at thecenter of the tissue where mineralization occurs through matrix vesicles(black arrows), and also depicts banded collagen I fibers with anapproximate diameter of 30 nm (white arrows) (scale bar=200 nm); FIG. 2c shows mineralization also occurring through intrafibrillarcalcification as seen by mineral crystals (arrows) along collagenfibrils (scale bar=200 nm); FIG. 2 d illustrates the edge of theengineering bone (EB) having a fibrous periostuem (FP) as indicated byfibroblasts (scale bar=4 mm); and FIG. 2 e is an expansion of the boxedarea of FIG. 2 d which shows that the collagen between the fibroblastsin the (FP) lacked mineral crystals (scale bar=300 nm);

FIG. 3 depicts a wide angle x-ray diffraction (WAXS) pattern of the EBCaccording to the present invention, showing that the mineral ishydroxyapatite when compared with peak locations and relativeintensities for the hydroxyapatite standard;

FIG. 4 a is a photograph of an EBC explant according to the presentinvention after a 4 week implantation (scale bar=5 mm);

FIGS. 4 b-c illustrate H and E stains of (b) an edge of a demineralizedexplant, wherein fibrous periosteum (FP) is seen on the edge of thesample, along with osteoblasts (arrow) differentiating fromosteoprogenitor cells of FP and invading engineered bone (EB), and (c)the center of a demineralized engineered bone which shows osteocytes(black arrows) and blood vessels (white arrows) (scale bar=62.5 mm);

FIGS. 4 d-e depict EBC explants stained for (d) calcification usingAlizarin red and (e) collagen I (scale bar=250 mm);

FIGS. 5 a-d illustrate TEM of an EBC explant according to the presentinvention, wherein FIG. 5 a depicts an edge of the EBC hasosteoprogenitor cells (black arrows) that are differentiating intoosteoblasts (white arrows) indicating periosteum function (scale bar=8mm); FIG. 5 b illustrates an osteocyte sitting in lacuna within the bonematrix (scale bar=2 mm); FIG. 5 c illustrates an osteoclast found inengineered bone resorbing demineralized bone matrix (BM) containsseveral nuclei (white arrows) and ruffled borders (black arrows),distinctions of this cell type (scale bar=10 mm); and FIG. 5 d is amagnification of the boxed area of FIG. 5 c which shows the ruffledborder in more detail, illustrating the empty (*) and filled (arrows)vacuoles (scale bar=1 mm);

FIGS. 6 a-c illustrate the fabrication of bone molds which may be usedfor formation of bone constructs with native bone dimensions accordingto the present invention, wherein FIG. 6 a depicts a rat femur sprayedwith silicone release and embedded in liquid silicone; FIG. 6 billustrates how, after the silicone has cured, the bone is cut form thesilicone, leaving a 3-D mold of native bone as shown in FIG. 6 c;

FIGS. 7 a-c illustrate a native adult rate medial collateral ligament(MCL) after (a) H & E staining; (b) immunostaining with collagen type I;and (c) immunostaining with elastin C;

FIGS. 8 a is a photograph of a three-dimensional ligament constructaccording to the present invention;

FIGS. 8 b-c depict H & E staining of a section taken from themid-section of a ligament construct according to the present inventionat two different magnifications;

FIG. 8 d depicts immunostaining of a mid-section of a ligament constructaccording to the present invention with collagen type I;

FIGS. 9 a-c are photographs of bone-ligament-bone (BLB) constructsengineered in an unchambered culture dish (a) and a chambered culturedish (b-c) in accordance with the present invention;

FIG. 10 a is a photographs of a BLB construct according to the presentinvention;

FIGS. 10 b-e are photographs of steps for MCL replacement according tothe present invention, wherein FIG. 10 b is a photograph after the MCLhas been removed and holes have been drilled for BLB implantation; FIG.10 c depicts a BLB placed inside silicone tubing and secured inreplacement of the excised MCL; FIG. 10 d depicts a BLB construct withinthe silicone tubing one month following implantation; and FIG. 10 edepicts the engineered BLB following one month implantation (black barin each photograph represents 5 mm length);

FIGS. 11 a-d illustrate the histology of BLB constructs according to thepresent invention explanted after one month following MCL replacement(sections were taken from the mid-section of the ligament portion of theexplanted construct), wherein FIGS. 11 a-b depict H & E staining of BLBconstructs at two different magnifications, and FIGS. 11 c-d depictimmunostaining of BLB constructs with collagen type I and elastin,respectively; and

FIGS. 12 a-c are graphs of nominal stress vs. nominal strain data forBLB constructs according to the present invention before (a) and after(b) one month implantation for MCL replacement, and data for native MCL(c).

DETAILED DESCRIPTION OF THE INVENTION

As required, detailed embodiments of the present invention are disclosedherein; however, it is to be understood that the disclosed embodimentsare merely exemplary of the invention that may be embodied in variousand alternative forms. The figures are not necessarily to scale, somefeatures may be exaggerated or minimized to show details of particularcomponents. Therefore, specific structural and functional detailsdisclosed herein are not to be interpreted as limiting, but merely as arepresentative basis for teaching one skilled in the art to variouslyemploy the present invention.

By way of example, the bone, ligament, and bone-ligament constructs andsystems and methods for their production according to the presentinvention are described with reference to the use of tissue harvestedfrom rats. However, it is fully contemplated that tissue from anymammal, including human beings, could be similarly utilized according tothe method described herein. The constructs, systems and methods of thepresent invention are not intended to be limited to one particular cellorigin or age, construct shape or dimensions, time frame, componentconcentration, or culture condition. One skilled in the art can readilyappreciate that various modifications can be made to the constructs,systems and methods described herein without departing from the scope ofthe invention disclosed.

Unless otherwise indicated, all solutions and media described herein maybe prepared and stored at 4 C before, and then warmed to 37 C in aheated water bath immediately before use. It is understood that allreagent measurements, materials, submersion times, and other valuesdescribed herein are approximate, and can be reasonably varied withoutaffecting the method and resulting constructs. Furthermore, theapproximate volumes of reagents within solutions described herein may bealtered to provide a solution with reagents having similar volumeratios.

According to a first aspect of the present invention, a system andmethod are provided for producing self-organized, 3D bone tissueconstructs solely from BMSC and their autogenous ECM, without the use ofartificial, exogenous scaffolding. The system and method describedherein bypass the many design challenges and limitations associated withthe engineering of composite bone constructs with exogenous scaffolding.As described below, the successful fabrication of engineered boneconstructs according to the present invention has been confirmed usinghistological and immunofluorescent markers for alkaline phosphataseactivity, type I collagen, osteocalcin, and absence of type II collagen.The engineered tissues have been further analyzed structurally usingtransmission electron microscopy (TEM) and functionally with tensiletests.

BMSC are multipotent, mesenchymal stem cells that can differentiate intobone, cartilage, ligament, adipose tissue, and muscle (Alhadlaq and Mao,Stem Cells Dev 13: 436, 2004; Pittenger and Martin, Circ Res 95: 9,2004) in response to chemical signals and generate and mineralize theirown autogenous ECM. BMSC can be easily isolated from autologous sourcesand therefore serve as an attractive candidate for tissue engineering.Since a specific bone marker does not exist, an engineered tissue ischaracterized as being bone by a number of criteria. The presence ofalkaline phosphatase, an enzyme that cleaves phosphate ions from organicmolecules, is a precursor to mineralization of bone and thus an earlysign of bone formation (Nauman et al., Calcif Tissue Int 73: 147, 2003).Bone is composed of predominantly type I collagen mineralized withhydroxyapatite crystals and contains osteocalcin, so the presence ofthese molecules further substantiates differentiation of BMSC towardsbone. The absence of type II collagen, the predominant protein ofcartilage, is often used to rule out differentiation to cartilage. Thestructural properties of bone including cells with an osteogenicmorphology and nucleation and growth of mineralized structures througheither matrix vesicles or intrafibrillar mineralization can be monitoredusing light and electron microscopy. The periosteum of bone is fibroustissue found on the bone surface that contains fibroblasts,osteoprogenitor cells, and unmineralized type I collagen.Osteoprogenitor cells differentiate into osteoblasts during bone growthand remodeling. The periosteum can be identified through microscopy dueto the elongated shape of fibroblasts and osteoprogenitor cells versusthe round presentation of osteoblasts. The presence of rows ofosteoblasts between the fibrous periosteum and the mineralized bone coreis an indication of a functioning periosteum since the osteoprogenitorcells are actively differentiating to osteoblasts.

According to the present invention, BMSC isolation and expansion may beaccomplished as follows. Under aseptic conditions, bone marrow may becollected from a bone of a host animal, such as the femur and tibia offemale Fisher 344 rats. The soft tissues of the leg are removed from thefemur and tibia, both ends of the bones are detached, and the marrowflushed out using a syringe (e.g., 25 gage needle) filled withDulbecco's Modified Eagle Medium (DMEM; Gibco, Rockville, Md.). Themarrow may be vortexed and then centrifuged at 480 g for ˜5 minutes,such as using a ThermaForma General Purpose Centrifuge. The pellet maybe resuspended in 10 ml growth medium (oGM), including DMEM with 20volume % fetal bovine serum (FBS; Gibco), 6 ng/ml basic fibroblastgrowth factor (bFGF; Peprotech, Rocky Hill, N.J.), 10⁻⁸ M dexamethasone(dex; Sigma-Aldrich, St. Louis, Mo.), and 1% antibiotic-antimycotic(Gibco), and plated into tissue culture dishes (e.g., 100 mm diameter).The dishes may be kept in an incubator at 37° C., 95% humidity, and 5%CO₂. After ˜48 h, the non-adherent cells may be removed by replacing theoGM with differentiation medium (oDM), further described below. Theadherent BMSC are cultured to 80% confluence, at which time cells areenzymatically removed from the plate using a 0.25% trypsin-EDTA solution(Gibco) and passaged. Cells may be plated onto construct dishes withinthe third to fifth passages.

Tissue culture plastic dishes (e.g., 35 mm in diameter; BD Biosciences)may be filled with 1.5 ml SYLGARD® (type 184 silicone elastomer; DowCorning Corp., Midland, Mich.) and serve as a substrate for constructformation. The polymer may be allowed to cure for approximately threeweeks before use. The SYLGARD® may be coated with 3 mg/cm² natural mouselaminin (Invitrogen, Carlsbad, Calif.) by filling dishes with 3 ml of a9.6 mg/ml laminin solution in Dulbecco's phosphate buffered saline(DPBS; GIBCO). The laminin solution may be evaporated overnight in abiosafety cabinet. Dishes are rinsed with DPBS and then filled with oneml of DMEM containing 20% FBS and 1% antibiotic-antimycotic. The dishesmay then be sterilized via exposure to ultraviolet radiation (e.g.,wavelength, 253.7 nm; bulb G30T8) in a biological safety cabinet for ˜60minutes, then kept in an incubator for 5-8 days prior to plating BMSC.

After the incubation, the medium is aspirated from the dish and BMSC maybe seeded onto each dish in 2 ml oGM supplemented with 0.13 mg/mlL-ascorbic acid-2-phosphate (asc-2-phos; Sigma-Aldrich) and 0.05 mg/mlL-proline (Sigma-Aldrich). According to one aspect of the presentinvention, this formulation may result in 30 dishes in total with aninitial cell density of 200,000 BMSC/dish. The cells may be fed oGMsupplemented with asc-2-phos and L-proline every 2 days until confluenceis reached. Once the cells reach confluence, two minuticn pins (e.g.,0.2 mm diameter and 1 cm long; Fine Science Tools, San Francisco,Calif.), may be pinned onto the cell monolayer in spaced relationship(e.g., 1.5 cm apart) to serve as anchors to constrain formation of theconstruct to a particular geometry. However, the pins are not requiredand do not constitute scaffolding as employed in prior art methods, asthey may be used to define the endpoints of the construct, but the BMSCare not disposed therewithin.

Bone formation in vitro from BMSC may utilize osteogenic media includingascorbic acid, dexamethasone (dex), and an organic phosphate. Ascorbicacid maintains connective tissue and regulates ATPase, alkalinephosphatase, and protein synthesis in cultures of osteoblasts. Dex, asynthetic glucocorticoid, stimulates osteoblastic and adipogenicdifferentiation from BMSC. Phosphates provide phosphate ions for matrixmineralization. In addition to these nutrients, basic fibroblast growthfactor (bFGF) and transforming growth factor beta (TGF-β) contribute tobone development in vitro but are not required for osteogenicdifferentiation from BMSC. bFGF, commonly used as a potent mitogen formany types of mesenchymal cells, increases mineralization, alkalinephosphatase activity, and the concentrations of bone specific markerssuch as calcium and osteocalcin when administered to BMSC in anosteogenic medium (Scutt and Bertram, Calcif Tissue Int 64: 69, 1999;Lisignoli et al, Biomaterials 22: 2095, 2001). TGF-β regulatesosteoblast replication and migration, increases alkaline phosphataseactivity, and stimulates collagen production and matrix maturation inbone cultures derived from both osteogenic cells and BMSC (Locklin etal., Cell Biol Int 23: 185, 1999).

At confluence, the oGM is switched to a second, differentiation medium(oDM) which may comprise DMEM with 7% horse serum (Gibco), 0.13 mg/mlasc-2-phos, 0.05 mg/ml L-proline, and 2 ng/ml transforming growth factorbeta (TGF-β; Peprotech) to induce construct formation. The oDM may bechanged every 2-3 days until the constructs are to be used.

Seven days after 3D construct formation, the engineered bone constructs(ESC) according to the present invention were mounted on a holder usingtissue freezing medium (Triangle Biomedical Sciences, Durham, N.C.) andimmersed in −80° C. isopentane. The frozen samples were slicedlongitudinally to a thickness of 9-12 mm using a Microm HM 500 cryostatsystem. The slides were then used for either histological staining forlight microscopy or immunofluorescent staining. For histochemicalstaining, tissue sections were fixed with methanol and stained foreither calcification with Alizarin Red or hematoxylin and eosin toobserve tissue structure. The remaining sections were fixed in acetoneand stained for alkaline phosphatase activity.

Immunofluorescent staining was performed to detect the presence ofcollagen I, collagen II, and osteocalcin. Frozen sections were fixedwith methanol for 5 min and rinsed 3 times with DPBS. Sections were thenblocked for 30 min with Ham's F-12 containing 5% donkey serum (DS;Jackson ImmunoResearch Labs, Inc, West Grove, Pa.) at 37° C. Sectionswere then incubated for 2 h with the primary antibodies in Ham's F-12containing 1% DS. The concentrations of each of the antibodies were asfollows: 5 mg/ml of rabbit anti-rat collagen I (Abcam Inc., Cambridge,Mass.), 5 mg/ml of mouse anti-rat collagen II (Calbiochem, Darmstadt,Germany), and 10 mg/ml of mouse anti-rat osteocalcin (Abeam). Sampleswere then rinsed 3 times with Ham's F-12 and were blocked again in Ham'sF-12 containing 5% DS at 37° C. for 10 min. The secondary antibodies (5mg/ml) were then applied to the sections for 1 h as follows: Alexa Fluor488 donkey anti-rabbit IgG (Molecular Probes, Eugene, Oreg.) forcollagen I, Alexa Fluor 555 donkey anti-mouse IgG (Molecular Probes) forcollagen II, and Alexa Fluor 488 donkey anti-mouse IgG (MolecularProbes) for osteocalcin. A Nikon Eclipse TS100 microscope equipped withan X-Cite 120 Fluorescence Illumination System was used to image thehistochemically and immunofluorescently marked sections. Controls wereperformed for the immunofluorescent staining (data not shown). Imageswere captured using a Diagnostic Instruments Spot Insight Color camera.

Samples were fixed for TEM in 2.5% glutaraldehyde (Electron MicroscopySciences, Hatfield, Pa.) in 0.1 M Sorensen's buffer, pH 7.4, for 24 h at4° C. Constructs were thoroughly rinsed with Sorenson's buffer, and postfixed with 1% osmium tetroxide in Sorensen's buffer for 2 h. Sampleswere then rinsed with double distilled water and stained with 8% uranylacetate in double distilled water for 1 h. Constructs were dehydrated ina graded series of ethanol, treated with propylene oxide, and embeddedin Epon 812. Longitudinal ultra-thin sections, 70 nm thick, wereprepared and stained with uranyl acetate and lead citrate. The sectionswere examined using a Philips CM100 electron microscope at 60 kV. Imageswere digitally recorded using a Hamamatsu ORCA-HR digital camera systemoperated using AMT software (Advanced Microscopy Techniques Corp.,Danvers, Mass.).

Tensile tests were performed on EBG engineered in vitro according to thepresent invention either 7 days (n=4) or 6 weeks (n=3) after 3Dconstruct formation. Tests were performed using a custom tabletoptensiometer attached to a Nikon SMZ1500 dissection microscope. Theconstructs were immersed into a bath of DPBS and the ends were graspedby clamps. The dimensions of the construct were measured prior to eachtest for a cross-sectional area calculation using a reticule in theeyepiece of the microscope. Beads 25 mm in diameter (Interactive MedicalTechnologies; Irvine, Calif.) were brushed on the surface of theconstruct as position markers for digital image correlation (DIC)analysis of tensile strain. Constructs were stretched at a strain rateof 0.01 s⁻¹. Force was measured using a custom force transducer andmonitored using LABVIEW software (National Instruments, Austin, Tex.);the resolution of the force transducer used for testing the EBC was 1mN. Images of the construct were captured using a Basler camera attachedto the Nikon dissection microscope at a frequency of 2.5 s⁻¹ during thetest to follow the location of the DIC markers. The bead position fromeach image was recorded using LABVIEW software with a resolution of 5 mmand length between beads was calculated by subtracting the position ofone bead from another. Nominal stress was calculated using the equationσ=F/A₀ where σ is nominal stress, F is force, and A₀ is the initialcross-sectional area of the sample. Nominal strain was calculated usingthe equation ε=(l−l_(o))/l_(o), where ε is the nominal strain, l is thecurrent length between beads, and l_(o) is the original length. Moduliwere obtained by calculating the slope of the tangent of stress versusstrain plots at the maximum strain prior to failure.

Seven days after 3D construct formation, EBC were placed into a piece ofsterile TYGON® silicone tubing (United States Plastics Corp., Lima,Ohio) that had inner and outer diameters of 1.6 and 3.2 mm,respectively, and a length of 1.5 cm. Fisher 344 rats were anesthetizedusing 0.001 ml Nembutal sodium solution (pentobarbital sodium injection;Ovation Pharmaceutical Inc., Deerfield, Ill.) per gram of the animal.The silicone tubes containing the constructs were then implanted betweenthe biceps femoris and the quadriceps of the left leg of the hostanimals. The silicone tubing was used in order to identify theengineered tissue during explantation.

Four weeks after implantation, constructs were removed from the animal.The diameter of the explants was measured using electronic calipers. Thesamples were then either frozen and mounted in tissue freezing mediumand stored at −80° C. or fixed in 2.5% glutaraldehyde and mounted inparaffin for histological studies.

Wide angle X-ray diffraction (WAXS) was performed on EBC (n=2) samplesaccording to the present invention 6 weeks in culture post 3D constructformation. The EBC samples were removed from their medium and allowed todry. The EBC samples were then placed on a single-crystal siliconsubstrate and examined with a Bruker D8 Discover diffractometer using CuKa radiation and a source voltage of 40 kV. The samples wereinvestigated over a 2q range of 23-55° with 20 counts recorded/degree.Scattering resulting from the substrate was subtracted and resultingpeaks were identified using the hydroxyapatite Powder Diffraction Filefrom the Joint Committee on Powder Diffraction Standards (JCPDS;Swarthmore, Pa.) (Hydroxyapatite Reference XRD peaks (ICDD:01-073-0293)).

As described above, in accordance with the system and method of thepresent invention, BMSC are cultured on construct dishes in a mediumthat induces osteogenic differentiation (oGM). Approximately 2-4 daysafter plating, the cells reach confluence and are switched to a secondmedium (oDM) containing a lower serum concentration and TGF-β, and twominutien pins are attached to the culture dish as constraint points.After a sufficient amount of ECM is produced, the tissue monolayer liftsfrom the substrate and contracts radially. The placement of the twoconstraint points inhibits full contraction and the monolayerself-organizes into a cylindrical tissue construct dependent on theplacement of any minutien pins which, according to one aspect of thepresent invention, may be about 1.5 cm in length and an elliptical crosssection with major and minor axes of 1.0±0.07 mm and 0.78±0.13 mm,respectively (FIG. 1 a). EBC formation according to the presentinvention is complete within ˜14 days of plating the BMSC onto the cellculture dishes. The resulting engineered tissue is completely solid,which suggests that the cells within the EBC remodel the tissue duringand after construct formation. The EBC were kept in culture either 7days or 6 weeks after formation of 3D structure prior to being fixed forhistological and structural characterization.

At 7 days post 3D EBC formation, hematoxylin and eosin (H and E)staining revealed that the self-assembled EBC were composed of bothdense and fibrous regions (FIG. 1 b). The bulk of the tissue stainedpositively for alkaline phosphatase activity (FIG. 1 c), and calciumdeposits were located throughout the samples as seen by Alizarin Redstaining (FIG. 1 d). The EBC according to the present invention stainedintensely throughout for type I collagen (FIG. 1 e) and lacked type IIcollagen (data not shown), which is consistent with the constitution ofnative bone. At this point, the EBC lacked osteocalcin (data not shown).TEM verified that the EBC contained osteoblasts (FIG. 2 a) in acollagenous matrix undergoing mineralization. The diameter of thecollagen was 29±4. nm which corresponds to the diameter of type Icollagen found in the femur (Ameye and Young, Glycobiology 12: 107R,2002). Mineralization was noted to occur through both matrix vesiclesand intrafibrillar calcification (FIGS. 2 b-c). The onset of periosteumdevelopment was observed by the presence of fibroblasts and axiallyaligned unmineralized type I collagen on the periphery of the EBC (FIGS.2 d-e). Wide angle X-ray scattering (WAXS) diffraction peak locationsfor the EBC corresponded with those of hydroxyapatite verifying that thecrystals seen in the TEM micrographs were indeed hydroxyapatite (FIG.3).

The EBC according to the present invention were able to maintain theirsize and shape after pins were removed from the edges. When theconstructs were grabbed by forceps at their centers, they were able toresist deformation under the pinching loads of the forceps, and the EBCresist compressive and bending deformation. Tension tests performed onEBC in culture at 7 days and 6 weeks post 3D EBC formation revealedmaximum tangent moduli of 7.5±0.5 MPa (n=4) and 29+9 MPa (n=3),respectively. The cross-sectional area remained the same between 7 daysand 6 weeks post 3D construct formation. The tangent moduli arenormalized with respect to the cross-sectional area of the EBC;therefore, the increase of stiffness between the two time points appearsto be a measure of EBC phenotype development, and not of physicalgrowth.

At 7 days post 3D construct formation, the EBC were implanted for 4weeks between the biceps femoris and quadriceps of Fisher 344 rats.While implanted, the EBC grew and remodeled so that the resultingexplant was cylindrical with a circular cross section. The diameter ofthe explant was 1.6±0.3 mm, equaling the inner diameter of the siliconetubing they were placed in during implantation (FIG. 4 a). H and Estaining of the demineralized explants revealed a structure thatappeared similar to that of native bone (FIGS. 4 b-c). Osteocytes inlacunae and blood vessels were seen throughout the construct (FIGS. 4b-c). Further development of a periosteum-like structure was seenfollowing implantation, as indicated by the fibrous tissue along theedge of the bone explants and the neighboring osteoblast-like cells(FIG. 4 b). Alizarin Red staining of explant sections showed increasedamounts of calcification (FIG. 4 d) when compared to those of theconstruct engineered in vitro (FIG. 1 d). The explanted constructscontained type I collagen (FIG. 4 e) and lacked type II collagen (datanot shown). After implantation, osteocalcin was also evenly distributedthroughout the bone explants (data not shown).

TEM of the demineralized explants showed a periosteum-like structurecontaining osteoprogenitor cells and rows of osteoblast-like cells (FIG.5 a). This structure is similar to that of native bone in whichosteoblasts differentiate from progenitor cells within the periosteum inrows and migrate towards the bone matrix. Osteocytes in lacunae wereseen within the bone matrix (FIG. 5 b). Osteoclasts were foundthroughout the mineralized tissue region (FIGS. 5 c-d). The osteoclastswere identified via their multinucleation (FIG. 5 c), ruffled borders,and a plethora of vacuoles used by the cell for storage of resorbedmaterial (FIG. 5 d).

Therefore, the system and method according to the present inventionproduce 3D bone constructs which self-organized in osteogenic media fromonly BMSC and their autogenous ECM, without disposing the cells withinan exogenous scaffold. The development of a periosteum-like structure inEBC in vitro as described herein has not been reported in 2D bonenodules or in bones engineered using exogenous scaffolding. The presenceof a periosteum-like structure is also significant since it indicatesboth fibrogenic and osteogenic differentiation from a single cell sourcewithin the same culture environment. This suggests that the BMSC weredifferentiating due to the cues delivered through the medium andpreferentially as a result of their location within the construct.

After 7 days post 3D construct formation, the EBC exhibit alkalinephosphatase activity and are composed of mineralized type I collagenenclosed within a fibrous periosteum-like tissue. The mechanicalproperties of the EBC according to the present invention improved overtime in vitro; tangent stiffness increased by a factor of four over afive-week period. No significant physical size change occurred in vitrofrom 7 days to 6 weeks in culture after 3D construct formation,indicating phenotype development due to tissue remodeling or increasedcollagen or mineral production. After 4 weeks in vivo, the EBC grew toequal the size of the tubing it was placed into prior to implantation,and the phenotype of the EBC according to the present inventioncontinued to advance during implantation in vivo. The explants containeda vascularized bone structure with osteoblasts, osteocytes andosteoclasts. The explants also contained osteocalcin which was notpresent before implantation and qualitatively stained more intensely formineralization. The explants also had a functional periosteum-liketissue containing osteoprogenitor cells that were undergoingdifferentiation to osteoblasts. The observation of osteoprogenitor cellsactively differentiating into osteoblasts as well as the presence ofosteoclasts demonstrates that the EBC actively grew and remodeled invivo in a manner similar to that of native bone.

The addition of bFGF to the culture medium according to the presentinvention may allow for monolayer formation rather than the formation ofbone nodules. The mitogenic effects of bFGF in addition to dex in thepresent invention may have increased proliferation, thus allowing formonolayer formation rather than the formation of nodules. TGF-β may bethe factor that controls 2D versus 3D construct formation. Although theoverall effects of this growth factor on BMSC are not fully known, it isgenerally used in culture to stimulate collagen production, matrixmaturation, and to induce chondrogenic differentiation from BMSC. TGF-βmay increase the rate of collagen production at an early stage of EBCdevelopment and prior to full osteogenic differentiation.

The tangent moduli of the EBC according to the present invention at 7days and 6 weeks in culture post 3D construct formation were 7.5±0.5 MPaand 29±9 MPa, respectively. Previous studies have reported the moduli ofunmineralized and mineralized embryonic bone as 1.11 MPa and 117 MPa,respectively (Tanck et al., Bone 35: 186, 2004). The mechanicalproperties of the EBC are consistent with developing native bone sincethe moduli lie within the limits of native embryonic bone. The tangentmodulus is a measure of constitutive stiffness and is independent ofconstruct size; the increase in EBC tangent moduli between 7 days and 6weeks in culture post 3D construct formation therefore may be anindication of phenotype advancement rather than physical growth.

The system and method of engineering bone-like structures according tothe present invention, in which cells are cultured to secrete, assemble,and mineralize their own three dimensional scaffolding, bypasses thecomplexity of engineering a scaffold and shifts the paradigm of bonetissue engineering to the guided self-assembly of an autogenousextracellular matrix. Furthermore, the system and method describedherein utilize the culture of BMSC that can be isolated from autologoussources without major ethical issues, and may be used clinically withminimal risk of rejection. The EBC according to the present inventionprovides a functional periosteum around an engineered bone, thus furtheradvancing the state of bone engineering. This system and method toengineer bone tissues can be used as a heuristic approach to tissueengineering for large bone defect repair or replacement without the useof an exogenous scaffold and as a model for bone formation throughintramembranous ossification.

These scaffold-less bone constructs not only contribute significantly tothe field of bone engineering, but also to the field of stem cellresearch. In the system and method described herein, osteogenic andfibroblastic differentiation of BMSC resulted within the same cultureenvironment, suggesting that stem cells may differentiate due tomechanical signals and cues from their location relative to other cellsin an engineered construct in addition to chemical signals. Mechanicallyconstraining the contractile monolayer induces tensile strain along themajor axis of the constructs and preferentially orients the collagenfibers in this direction. The constructs according to the presentinvention are the first 3D, scaffold-less tissues developed from BMSCthat demonstrate phenotype advancement in vitro and in vivo.

An additional goal of tissue engineering is to fabricate tissue for usein repair of tissue damaged as a result of disease, trauma and surgery,especially for cases where the amount of bone loss creates a criticaldefect which will not repair by normal self-repair processes and needsintervention. In further accordance with the present invention,utilization of the EBC to repair a critical bone defect may produce anew bone segment in which the engineered construct will incorporate intothe host bone and form a viable interface that will restore thefunctionality.

According to another aspect of the present invention, using multipleEBC, larger, more complex bone shapes may be fabricated such as, but notlimited to, a femur or mandible bone in a rat. Multiple bone constructsmay be arranged laterally or longitudinally with respect to one anotherduring in vivo implantation and this arrangement may allow theconstructs to grow and remodel into a cohesive construct. The presentinvention further contemplates the use of imaging technologies tovisualize a healthy contralateral bone, generate a three-dimensionalmold (e.g., silicone) from that image, obtain BMSC from the patient(e.g., from the pelvic bone) needing the tissue replacement, andfabricate an engineered bone for replacement from the patient's ownBMSC.

Bone molds may be constructed according to the present invention bysubmerging native rat bones (e.g., femur, tibia, pelvis, mandible) intosilicone as it cures (FIG. 6). The bones may be dissected out of the ratand, after thoroughly drying, the bones may be sprayed with siliconemold release and suspended in liquid SYLGARD®. The SYLGARD® may beallowed to cure for approximately one week. The native bone may then beremoved from the silicone, leaving a mold in the shape of native bone.

Bone monolayers may be fabricated as described herein. Approximately oneweek after shifting to oDM, when the forming bone constructs have liftedfrom the substrate and have started to roll up (e.g., ˜20%), theconstructs may be implanted into molds and then into the host animals.In one example, a plurality of forming bone constructs (e.g., three) maybe placed into a mold. The constructs may be secured together, such asby suturing, the ends of the constructs may be secured to the mold, andthe mold may be secured inside the host animal to prevent migration ofthe mold within the host. In preliminary experiments utilizing thesystem and method according to the present invention, suturing threeconstructs together prior to implantation visually yielded one fusedconstruct.

In further accordance with the present invention, BMSC may be used tofabricate 3D scaffold-less ligament and bone-ligament constructs. Theability of BMSCs to differentiate into a specific lineage in vitro maybe controlled by the culture environment, differentiation-inducingagents, growth factors and mechanical stimulation. Previous attemptshave been made to create 3D ligaments in vitro using artificialscaffolding, but these have met with limited success because of thedifficulty in creating a construct that is biologically compatible withthe in vivo environment and integrates with bone tissue.

As a matter of background, mature ligament obtained from rat medialcollateral ligament (MCL) shows the characteristic wavy pattern ofcollagen fibrils with elongated cell nuclei dispersed amongst thefibrils (FIG. 7 a). Immunohistochemical staining of longitudinalsections of native MCL reveals positive staining for collagen I (FIG. 7b) and elastin (FIG. 7 c), and negative staining for collagen II (imagenot shown).

According to the present invention, self-organized 3-D ligamentconstructs (ELC) may be engineered from BMSC in fibrogenic media. Withreference to the system and method for producing EBC described above,for ligament the growth medium (fGM) includes Fetal Bovine Serum (FBS,Gibco BRL Cat#10437-028), 6 ng/ml basic fibroblast growth factor (bFGF;Peprotech, Rocky Hill, N.J.), 0.13 mg/ml asc-2-phos, 0.05 mg/mlL-proline, 5 ml A9909 (Sigma A9909), and differentiation medium (fDM)includes 460 ml DMEM with 35 ml 100% Horse Serum Albumin (HSA, Gibco BRLCat#16050-122), 0.13 mg/ml asc-2-phos, 0.05 mg/ml L-proline, 2 ng/mltransforming growth factor beta (TGF-β; Peprotech), and 5 ml A9909(Sigma A9909). Once the cells become confluent, pins may be inserted inthe substrate in spaced relationship to guide and constrain theresulting construct geometry.

As described above, since BMSC are multipotent cells that candifferentiate into a plurality of tissue types, several markers are usedto identify tissues in the developing constructs according to thepresent invention. Type I collagen, fibronectin and elastinimmunostaining are used as markers of ligament development. Staining fortype II collagen, aggrecan and tenascin-C are used as markers of thedeveloping interface between bone and ligament (enthesis). Morphologicalobservations of cellular and ECM structures using light and electronmicroscopy may be used to identify the presence of the expected cell andtissue types in the developing constructs.

With reference to FIG. 8 a, approximately 3 days after detachment of themonolayer, the cells self organized into a cylinder, wherein the lengthof the construct was determined by the placement of the pins (e.g., 15mm) and, the diameter in the image shown was approximately 500 μm. Invitro engineered ligament according to the present invention has anouter perimeter that looks similar to native ligament with highlyorganized collagen and elongated nuclei between fibril (FIGS. 8 b-c).Immunohistochemistry of the ELC indicates that the fibrillar perimeteris type I collagen (FIG. 8 d). In contrast to the ECM formed by the BMSCin osteogenic media, the engineered ligament ECM lacks mineralization asdetermined by an absence of alizarin red staining, does not stain foralkaline phosphatase activity, and lacks type II collagen (negativestaining data not shown). The tensile response of the engineeredligament is non-linear and viscoelastic; the response to cyclic loadingshows strain softening. The as-formed engineered ligaments have atangent stiffness of 2.8±1.8 MPa (n=3). After four weeks ofimplantation, the ligament explants grew physically in size and theirtangent stiffness increased by about an order of magnitude to 15.4±5.6MPa (n=3). Native adult rat MCL has a tangent stiffness of 550±50 MPa(n=2).

Preliminary mechanical tests of the ELC according to the presentinvention show that the constructs have the same nonlinearporoviscoelastic characteristics of native MCL. The use of exogenousscaffolds as in prior art methods often results in mechanical functionthat does not include these characteristics, whereas the constructsaccording to the present invention have the organized extra-cellularmatrix proteins, proteoglycans and fluid phases of native tissue.

To be most useful for tissue replacement, the ELCs need an anchoringmechanism that will allow functional attachment to the bone in thelocation of the ligament replacement. Therefore, an engineered ligamentwith engineered bone at each end would be optimal for repair if theengineered bone integrates with the native bone in vivo followingimplantation. According to the present invention, a system and methodare provided for co-culturing self-organized 3D ligament tissue fromBMSC with engineered scaffold-less bone tissue to formbone-ligament-bone (BLB) constructs having a mechanically viableenthesis to withstand the transmission of physiological strains.

BLB construct formation according to the present invention may beaccomplished in different ways. According to one aspect of the presentinvention, the fGM may be removed from previously preparedlaminin-coated plates and 2 ml of a cell suspension containing 2×10⁵cells per ml of fGM may be plated in each culture dish and placed in a37° C. 5% CO₂ incubator, wherein the medium may be changed every 2-3days. After the cells become confluent, approximately 3 days later,engineered bone tissue fabricated as described above and having somedegree of confluence may be cut into segments (e.g., 5 mm in length) andplaced in contact with the cell monolayer. According to one aspect ofthe present invention, the bone constructs may be pinned (e.g., usingtwo minutien pins) on top of the cell monolayer facing each other sothat the inner ends are spaced apart (e.g., 1 cm). At this point, thefGM may be replaced by fDM. The ligament monolayer is allowed to roll upand at least partially surround each of the bone monolayers, creating afunctional integration of the bone with the ligament and forming a 3-D,self-organized BLB construct (FIG. 9 a). Of course, it is understoodthat a bone-ligament construct having bone at only one end is also fullycontemplated according to the present invention.

The effect of the ligament DM (fDM) on continued bone formation isunknown. Preliminary data suggest that the bony ends of the co-culturecontinue to develop bone matrix and produce osteocytes once the boneshave formed, even if the medium is no longer supplemented with dex. Achambered cell culture dish (FIG. 9 b) may be utilized to allow bathingof the ligament portion of the construct in fDM while exposing the boneportions of the construct in bone DM (oDM). At the time of ELCformation, it is unknown if osteogenic precursor cells remain (FIG. 9c). If bone precursor cells do exist, in further accordance with thepresent invention, a fully formed ligament construct may be placed intoa chambered culture dish with the interior chamber exposed to fDM andthe outer chambers to oDM to create a BLB construct in this manner.

One of the most common sites for ligament damage and thus need forrepair is the anterior cruciate ligament (ACL). Due to the poor healingcapacity of the ACL following injury, surgical “reconstructions” orreplacements of the ligament, involving bone/ligament autografts orallografts, are performed at a rate of 400,000 per year. In allreplacement methodologies, the utilization of screws to fix the bonyplug of the graft to the native bone is not a permanent solution. Immunerejection or physical dislodging of the screw means a second surgicalintervention to reattach the graft. The accessibility of the ACLespecially in small animal models such as the rat limits the usefulnessof the ACL for studies of ligament replacement. Therefore, the MCL hasbeen used herein as a model to demonstrate the ability of the engineeredBLB constructs according to the present invention to incorporate intoendogenous bone tissue, and to grow and remodel in vitro and in vivo.Utilization of the BLB as an MCL replacement may result in incorporationof the bone segment of the BLB into the host bone and formation of aviable connection that will restore the functionality of the knee joint.

A representative example of a 3D BLB construct fabricated according tothe present invention is shown in FIG. 10 a, wherein the constructlength in this case is 15 mm with a width of 1 mm, although it isunderstood that BLB construct dimensions are not limited by thisexample. Five days following 3-D formation, the BLB construct was usedto replace the MCL in a rat. During implantation, the native MCL wasexcised, holes were drilled at the place of enthesis in both the femurand tibia (FIG. 10 b), a BLB was inserted into a silicone tube forsubsequent identification during explanation, and secured into placewith 7-0 suture (FIG. 10 c). Alternatively, the bone sections of theconstruct may be secured by suturing the construct to the surroundingconnective tissue. One month later, the entire knee was extracted fromthe animal (FIG. 10 d), the engineered construct was isolated fromsurrounding tissues, and the patellar, ACL, PCL, and LCL were excisedleaving the BLB-based MCL replacement tissue adhered to the femur andtibia (FIG. 10 e). Following 1 month implantation, the BLB constructfused with the bone at both the femur and tibia and increased in size,stiffness and strength, and the native MCL has been replaced by theremodeled BLB construct. The bones were then anchored into a tensiometerand the mechanical properties of the explanted BLB were measured (FIG.12).

Longitudinal sections were taken through the mid section of theBLB-based MCL replacement explant and stained with H and E. FIG. 11reveals collagen fibrils filling the entire cross-section of theexplant, while the collagen content resembles that of adult MCL, thereare many more nuclei present amongst the fibrils (FIGS. 11 a-b).Immunohistochemical staining of the sections shows positive staining forboth collagen I (FIG. 11 c) and elastin (FIG. 11 d) similar to thestaining pattern observed in native adult MCL (FIG. 7).

Using a custom-designed and built tensiometer for soft tissue testingthat includes digital image correlation measurements of sequential localtissue positions to accurately calculate actual tissue strain, severalload-unload cycles were conducted to examine the viscoelastic responseof in-vitro BLB constructs (FIG. 12 a), BLB explants (FIG. 12 b) andnative adult rat MCL (FIG. 12 c). The tangent stiffness is computed asthe slope of the first load cycle at a strain of 0.06. The in vitroconstructs have a ligament tangent stiffness of 2.8±1.8 MPa (n=3). Thephenotype development within the ligament region of the BLB during onemonth of implantation is seen by comparing FIGS. 12 a and 12 b. The toeregion in the in vitro BLB construct (FIG. 12 a) becomes less pronouncedduring implantation (FIG. 12 b). The cyclic response shows theengineered tissue in vitro is non-linear and viscoelastic withhysteresis in each load-unload cycle (FIG. 12 a). The BLB retains thesesame characteristics as its mechanical stiffness increases by about anorder of magnitude during one month of implantation (FIGS. 12 b) to15.4±5.6 MPa (n=3). The native adult MCL exhibits the same mechanicalresponse to cyclic loading and is roughly an order of magnitude stiffer(550±50 MPa (n=2)) than the explanted BLB constructs (FIG. 12 c).

Therefore, the co-culture of engineered ligament and bone tissuesaccording to the present invention generates 3D cylindrical BLBconstructs with the morphological characteristics of bone and ligamentin vivo and a mechanically viable enthesis that structurally andbiochemically resembles that of neonatal tissue. The creation of anengineered bone/ligament co-culture with a viable interface in vitrogreatly expands the potential for ligament repair by providing afunctional enthesis to bone. In vivo implantation of EBCs and BLBsaccording to the present invention may further advance the phenotype andfunctionality of the bone, ligament and enthesis, and these engineeredconstructs may be viable treatments for tissue repair or replacement.The fabrication of engineered BLB constructs according to the presentinvention with the capability for growth and remodeling of the ligamentand permanent incorporation of the engineered bone into the surgicalsite may vastly improve the lifespan of the surgical repair, forexample, of a damaged ACL.

The development of a BLB construct as in the present invention that hasa structurally sound enthesis offers innumerable implications for futureinvestigations on bone and ligament development. For example, the BLBconstruct may be used to investigate basic science questions such as theeffects of various growth factors—dexamethasone, basic fibroblast growthfactor, transforming growth factor beta, bone morphological proteins,etc.—on osteogenesis, fibrogenesis and enthesis formation. The BLBconstruct may also be used to investigate the effect of geneticmanipulation via null vs. over-expression of specific cytoskeletalproteins or osteogenic regulatory factors on the bone development, or toinvestigate the effects of various mechanical loading protocols in vitroand in vivo on bone and ligament development.

While embodiments of the invention have been illustrated and described,it is not intended that these embodiments illustrate and describe allpossible forms of the invention. Rather, the words used in thespecification are words of description rather than limitation, and it isunderstood that various changes may be made without departing from thespirit and scope of the invention.

What is claimed is:
 1. A system for forming a bone construct,comprising: a substrate; and bone marrow stromal cells provided on thesubstrate without disposing the cells within an exogenous scaffold, thecells cultured in vitro in osteogenic media such that the cells form aconfluent monolayer and detach from the substrate to form aself-organized three-dimensional bone construct.
 2. The system accordingto claim 1, further comprising at least two anchors secured to thesubstrate and the confluent monolayer in spaced relationship toconstrain development of the three-dimensional bone construct.
 3. Thesystem according to claim 1, wherein the osteogenic media comprises abone growth medium and a bone differentiation medium.
 4. The systemaccording to claim 3, wherein the bone differentiation medium includes alower serum concentration compared with the bone growth medium.
 5. Thesystem according to claim 3, wherein the bone growth medium includesdexamethasone.
 6. The system according to claim 3, wherein the bonegrowth medium includes basic fibroblast growth factor.
 7. The systemaccording to claim 3, wherein the bone differentiation medium includestransforming growth factor beta.
 8. The system according to claim 1,further comprising a mold having a native bone shape into which at leastone bone construct is placed.
 9. A method for forming a bone construct,comprising: providing bone marrow stromal cells on a substrate withoutdisposing the cells within an exogenous scaffold; and culturing thecells in vitro in osteogenic media such that the cells form a confluentmonolayer and detach from the substrate to form a self-organizedthree-dimensional bone construct.
 10. The method according to claim 9,further comprising securing at least two anchors to the substrate andthe confluent monolayer in spaced relationship to constrain developmentof the three-dimensional bone construct.
 11. The method according toclaim 9, wherein the osteogenic media includes a bone growth medium anda bone differentiation medium, further comprising providing the cells onthe substrate in the bone growth medium, and switching to the bonedifferentiation medium once the cells form the confluent monolayer. 12.The method according to claim 11, wherein the bone differentiationmedium includes a lower serum concentration compared with the bonegrowth medium.
 13. The method according to claim 11, wherein the bonegrowth medium includes dexamethasone.
 14. The method according to claim11, wherein the bone growth medium includes basic fibroblast growthfactor.
 15. The method according to claim 11, wherein the bonedifferentiation medium includes transforming growth factor beta.
 16. Themethod according to claim 9, further comprising placing at least oneforming bone construct in a mold having a native bone shape.
 17. Themethod according to claim 16, wherein placing at least one forming boneconstruct includes securing a plurality of forming bone constructstogether.
 18. A bone construct, comprising: bone marrow stromal cellsprovided on a substrate without disposing the cells within an exogenousscaffold, the cells cultured in vitro in osteogenic media such that thecells form a confluent monolayer and detach from the substrate to form aself-organized three-dimensional bone construct.
 19. The bone constructaccording to claim 18, wherein the bone construct exhibitsmineralization and alkaline phosphatase activity, includes type Icollagen, and lacks type II collagen.
 20. A system for forming aligament construct, comprising: a substrate; and bone marrow stromalcells provided on the substrate without disposing the cells within anexogenous scaffold, the cells cultured in vitro in fibrogenic media suchthat the cells form a confluent monolayer and detach from the substrateto form a self-organized three-dimensional ligament construct.
 21. Thesystem according to claim 20, further comprising at least two anchorssecured to the substrate and the confluent monolayer to constraindevelopment of the three-dimensional ligament construct.
 22. The systemaccording to claim 20, wherein the fibrogenic media comprises a ligamentgrowth medium and a ligament differentiation medium.
 23. The systemaccording to claim 22, wherein the ligament differentiation mediumincludes a lower serum concentration compared with the ligament growthmedium.
 24. The system according to claim 22, wherein the ligamentgrowth medium includes basic fibroblast growth factor.
 25. The systemaccording to claim 22, wherein the ligament differentiation mediumincludes transforming growth factor beta.
 26. The system according toclaim 20, wherein the substrate is divided into two chambers, a firstchamber including the fibrogenic media to induce ligament formation, anda second chamber including osteogenic media to induce bone formation.27. A method for forming a ligament construct, comprising: providingbone marrow stromal cells on a substrate without disposing the cellswithin an exogenous scaffold; and culturing the cells in vitro infibrogenic media such that the cells form a confluent monolayer anddetach from the substrate to form a self-organized three-dimensionalligament construct.
 28. The method according to claim 27, furthercomprising securing at least two anchors to the substrate and theconfluent monolayer in spaced relationship to constrain development ofthe three-dimensional ligament construct.
 29. The method according toclaim 27, wherein the fibrogenic media includes a ligament growth mediumand a ligament differentiation medium, further comprising providing thecells on the substrate in the ligament growth medium, and switching tothe ligament differentiation medium once the cells form the confluentmonolayer.
 30. The method according to claim 29, wherein the ligamentdifferentiation medium includes a lower serum concentration comparedwith the ligament growth medium.
 31. The method according to claim 29,wherein the ligament growth medium includes basic fibroblast growthfactor.
 32. The method according to claim 29, wherein the ligamentdifferentiation medium includes transforming growth factor beta.
 33. Aligament construct, comprising: bone marrow stromal cells provided on asubstrate without disposing the cells within an exogenous scaffold, thecells cultured in vitro in fibrogenic media such that the cells form aconfluent monolayer and detach from the substrate to form aself-organized three-dimensional ligament construct.
 34. The ligamentconstruct according to claim 33, wherein the ligament construct lacksmineralization and alkaline phosphatase activity, includes type Icollagen, and lacks type II collagen.
 35. A system for forming abone-ligament construct, comprising: a substrate; bone marrow stromalcells provided on the substrate without disposing the cells within anexogenous scaffold, the cells cultured in vitro in fibrogenic media suchthat the cells form a confluent ligament monolayer; and at least onebone construct provided in contact with the confluent monolayer suchthat the monolayer detaches from the substrate to at least partiallysurround the at least one bone construct and functionally integratetherewith, thereby forming a three-dimensional bone-ligament construct.36. The system according to claim 35, wherein the at least one boneconstruct includes two spaced apart bone constructs secured to theconfluent monolayer to form a three-dimensional bone-ligament-boneconstruct.
 37. The system according to claim 35, wherein the substrateis divided into two chambers, a first chamber including the ligamentmonolayer and the fibrogenic media, and a second chamber including theat least one bone construct and osteogenic media.
 38. A method forforming a bone-ligament construct, comprising: providing bone marrowstromal cells on a substrate without disposing the cells in an exogenousscaffold; culturing the cells in vitro in fibrogenic media such that thecells form a confluent ligament monolayer; and providing at least onebone construct in contact with the confluent monolayer such that themonolayer detaches from the substrate to at least partially surround theat least one bone construct and functionally integrate therewith,thereby forming a three-dimensional bone-ligament construct.